PROJECT SUMMARY/ABSTRACT Hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in people under 30. Clinically, HCM is characterized by hyper-contractility and a thickened ventricular wall with severity that directly depends on the ratio of mutant to wild type protein expression, a concept known as allelic imbalance. HCM is most commonly associated with mutations in genes encoding the sarcomeric proteins ?-cardiac myosin and cardiac myosin-binding protein C (cMyBP-C). ?-cardiac myosin is a motor protein that assembles into thick filaments and coverts chemical energy from ATP into a mechanical force-generating lever arm swing required for muscle contraction. cMyBP-C is a long multi-modular structural protein that is thought to inhibit the actin- myosin interaction and mitigate the effects of calcium, regulating muscle contraction. While it is clear that ?- cardiac myosin and cMyBP-C are crucial for normal sarcomeric function, it is less clear how mutations in these proteins produce the severe hyper-contractile phenotype seen in HCM. The central goal of this training proposal is assess the impacts of HCM mutations on sarcomeric interactions and the overall ensemble phenotype. Despite the identification of over 700 HCM mutations in ?-cardiac myosin and cMyBP-C combined, there has been little success in linking genotype to disease phenotype. Due to mechanistic uncertainty, no small molecule therapies for HCM exist and treatment remains palliative. The difficulty in characterizing HCM can be partially attributed to lack of available technologies to study these highly organized proteins on the sarcomeric level. Single-molecule studies do not account for inter-motor interference in the motor ensemble and existing in-vitro assays are limited by variability and heterogeneity of the motility surface. The Sivaramakrishnan lab has therefore developed and obtained preliminary data documenting the utility of a DNA nanotube scaffold as a synthetic thick filament. Myosin and cMyBP-C can be patterned onto the DNA nanotube at precise intervals, recapitulating the native sarcomeric interactions. I propose to use DNA nanotube technology to test my central hypothesis through two aims. First, I will determine the impact of allelic imbalance on the overall ensemble phenotype by patterning varying ratios of HCM mutant and wild type onto a nanotube. Second, I will use the synthetic thick filament to dissect the contributions of cMyBP-C interactions and altered calcium effects in the hypercontractile phenotype of HCM. The findings from these experiments will substantially contribute to our understanding of how genotype translates to HCM phenotype, and will aid in the development of targeted pharmaceuticals.